Developmental and epileptic encephalopathies (DEEs) are severe epilepsy syndromes that manifest in infancy or early childhood and are characterized by intractable seizures, neurological and behavioral deficits, and a high risk of Sudden Unexpected Death in Epilepsy (SUDEP). While most DEEs are linked to variants in genes encoding ion channels, especially that of voltage-gated sodium (NaV) and potassium (KV) channels, DEE-linked variants in non-ion-channel genes may provide important insights into the etiology of disease. Recently, whole exome sequencing of DEE patients by our Peking University colleagues in China identified variants of uncertain significance (VUS) in ANK3, suggesting that deficits in ion channel localization may contribute to disease mechanisms. A large body of literature has shown that ankyrin-G, encoded by the ANK3 gene, plays a fundamental role in the localization of voltage-gated ion channels to critical neuronal plasma membrane subdomains, including the axon initial segment (AIS) and nodes of Ranvier, which are the sites of action potential (AP) initiation and propagation, respectively. Recently, we have discovered novel functions for ankyrin-G in the regulation of inhibitory synapses and control of neuronal excitability. However, the mechanisms underlying the link between ANK3 and epilepsy are incompletely understood. The long-term goal of our work is to understand how ankyrin-G dysfunction contributes to the etiology of neurological disorders, like DEE. The objective of this application is to use a knockout and rescue strategy to understand the cellular and electrophysiological effects of variants identified in the Peking University DEE cohort. Our central hypothesis is that DEE-associated ANK3 variants affect ankyrin-G function in controlling localization and function of voltage- and ligand-gated ion channels resulting in pyramidal cell dysfunction, contributing to the pathophysiology of DEE. We will test our hypothesis by pursuing two Specific Aims: 1) To understand the effects of human DEE-associated ANK3 variants on ankyrin- G-mediated ion channel localization. 2) To determine the electrophysiological consequences of ANK3 DEE- associated variants. The mechanisms underlying the link between ANK3 and epilepsy are incompletely understood, yet the recently discovered VUS from the DEE patient cohort in China suggest a novel DEE mechanism. The results of this work will have an important positive impact on the understanding of how ankyrin- G regulates neuronal excitability and how ankyrin-G loss-of-function contributes to complex neurological disorders, such as DEE. Most of the published work investigating DEE mechanisms has focused on patient variants within ion channel genes. However, treatment strategies targeted against perturbations in ion channel function have proven ineffective for many DEEs. Our approach may yield novel targets, which could serve as a guide to develop innovative therapeutic strategies to treat DEE and possibly other idiopathic forms of epilepsy. In addition, the results of these studies may uncover previously unappreciated functions of AnkG in neurotransmission.